A quick and simple method has been devised to permit the microscale synthesis of [alpha-32P]-AZT which is valuable as a probe of the mechanism of action of AZT. This approach may also be useful for application to related dideoxynucleoside inhibitors of HIV. By the combination of a variety of synthetic techniques, a novel analogue of AZT 5'-triphosphate has been prepared; namely, the beta, gamma-difluoromethylenephosphonate analogue. This material possesses a beta-gamma bond that cannot be hydrolyzed, and its pK(a) is similar to the parent AZT triphosphate. This analogue was found to be 30-fold less potent as an HIV reverse transcriptase inhibitor than AZT triphosphate, but it was 10 times more effective than the corresponding phosphonate analogue without the fluoro substitution, suggesting that this substitution provides a closer match than the oxygen bridge of the parent compound, AZT triphosphate.